The Toxicologist - Society of Toxicology

UGT1A gene induction, while this response was lost in hUGT1/Pxr -/- mice. In conclusion,
PXR mediates the gestational upregulation of the human UGT1A genes,
which may be driven by elevated glucocorticoid levels during late gestation.
(Supported by USPHS Grant GM086713 and ES010337)
386 BREAST MILK SUPPRESSES UDP-
GLUCURONOSYLTRANSFERASE 1A1 GENE
EXPRESSION IN THE GASTROINTESTINAL TRACT
AND INCREASES THE RISK FOR SEVERE
HYPERBILIRUBINEMIA AND BRAIN DAMAGE.
R. Fujiwara, S. Chen and R. H. Tukey. Pharmacology, University of California,
San Diego, La Jolla, CA.
Premature and term newborns are susceptible to physiological jaundice, which can
lead to acute bilirubin encephalopathy or the more permanent clinical sequelae of
bilirubin-induced neurological dysfunction (BIND). Accumulating evidence indicates
that breast-fed infants have a higher risk for developing BIND than formulafed
infants. However, the underlying mechanism linking severe hyperbilirubinemia
to breast feeding has not yet been elucidated. Here we describe a new mechanism
that places breast-fed neonates at risk for developing BIND. Humanized UDP-glucuronosyltransferase
1 (hUGT1) mice (Fujiwara R, et al. PNAS 107:5024-9, 2010)
develop severe hyperbilirubinemia during neonatal development. The steady-state
levels of total serum bilirubin (TSB) during development are concordant with expression
of UGT1A1 in the gastrointestinal tract. Approximately 10% of neonatal
hUGT1 mice develop BIND, characterized by seizures and the deposition of bilirubin
in the brain. In contrast, formula-fed hUGT1 mice have significantly lower levels
of TSB and do not develop seizures. Compared to breast-fed neonates, formulafed
hUGT1 mice have significantly induced levels of UGT1A1 in the GI tract.
Formula feeding has no effect on UGT1A1 gene expression in liver. The induction
of UGT1A1 by formula may be controlled by xenobiotic receptors PXR and CAR,
since the Cyp3a11 and Cyp2b10 genes are also induced. This data indicates that GI
tract control of UGT1A1 gene expression in newborns is sufficient in preventing
developmentally induced hyperbilirubinemia. As we define the role of breast milk
in regulating UGT1A1 gene expression, hUGT1 mice may prove useful as a screening
tool in examining nutritional supplements to aid in halting the progression of
hyperbilirubinemia. (Supported by USPHS grants GM086713 and ES010337)
387 TRANSCRIPTOMIC ANALYSIS REVEALS MECHANISM
UNDERLYING THE OFF-TARGET EFFECT OF THE
CHOLESTERYL-ESTER TRANSFER PROTEIN
INHIBITOR TORCETRAPIB.
J. Hoflack, N. Flint, M. Haiker, A. Stauffer, L. Suter-Dick, A. Roth, T. Weiser,
T. Singer, L. Müller, E. J. Niesor and R. G. Clerc. F Hoffmann La Roche, Basel,
Switzerland.
The development of the cholesteryl-ester transfer protein (CETP) inhibitor
Torcetrapib failed in a phase III clinical study. An effect on the aldosterone pathway
was suggested as responsible for the increased mortality. We therefore evaluated the
impact of Torcetrapib and of Angiotensin II on the transcriptome of the human aldosterone-producing
adrenocortical cell line H295R using microarrays and qRT-
PCR. Several key genes involved in adrenal-dependent steroidogenesis were robustly
regulated by Torcetrapib at concentrations as low as 1 nM. Torcetrapib
essentially elicited a transcriptomic pattern similar to that of Angiotensin II on the
aldosteroidogenesis pathway, but displayed also distinct features. In particular, upregulation
of the L-type Ca2+ channel alpha1c mRNA by Torcetrapib at the top of
the pathway, which was not observed with Angiotensin II, characterizes the different
properties of the two aldosteroidogenic agents. Consistently, inhibition of
Torcetrapib-dependent aldosteroidogenesis by the L-type-specific Ca2+ channel inhibitor
Nifedipine, not of the Angiotensin II-dependent aldosteroidogenesis, as
well as inhibition of aldosteroidogenic gene regulations occuring in response to
Torcetrapib, underscored the specific contribution of L-type Ca2+ channels in the
signaling cascade of Torcetrapib. This Torcetrapib-specific effect occurs at the
mRNA level to a wide extent and unravels a new activation of the signaling cascade
towards aldosterone production, through L-type Ca2+ channels. Dalcetrapib, a
CETP-targeting compound from a chemical class different from Torcetrapib, had
no observable effect on either aldosterone production nor on aldosteroidogenic
gene regulations up to 10 μM under identical experimental conditions. These transcriptome
data, confirmed later using various functional endpoints, first indicated
that the Torcetrapib-effect is not an on-target but a chemical class-specific, off-target
effect.
388 TRANSCRIPTIONAL INDUCTION OF GLUTATHION S-
TRANSFERASE BY CHLORPYRIFOS IN HEPG2 CELLS.
I. M. Medina 1 , M. C. Martinez 1 , M. Rubio 1 , A. E. Rojas 1 , M. L. Robledo 1 , M.
I. Giron 1 and G. Elizondo 2 . 1 Secretaria de Investigacion y Posgrado, Universidad
Autonoma de Nayarit, Tepic, Nayarit, Mexico and 2 bDepartamento de Biología
Celular, Centro de Investigación y de Estudios Avanzados del IPN, Mexico, DF,
Mexico. Sponsor: B. Quintanilla.
Organophosphates pesticides (OPs), such as chlorpyrifos, are the most widely used
insecticides worldwide. As a result of their extensive use, large populations of humans
are potentially expose to low doses of OPs, mainly due to the presence of
these compounds in food and drinking water. Glutathion S-transferase (GST) catalyzes
the nucleophilic attack of the tripeptide glutathione (GSH) on electrophilic
substrates, thus forming an important line of defense and protecting cell components
from reactive molecules. The action of GST on organophosphate pesticides
can lead to activation or detoxification. The aim of this study was to test the hypothesis
that OPs pesticides (chlorpyrifos) modulate the expression of GST alpha
(GSTA) and theta (GSTT) gene in HepG2 cells. To determine the expression of
GSTA and GSTT, quantitative real-time PCR assay (rtPCR) of the transcripts was
performed with gene-specific fluorescent labeled probes and the activity assays was
determined using the GST Assay Kit. Treatment with chlorpyrifos resulted in an induction
of GSTT and GSTA mRNA and activity relative to a control (untreated
cell cultures). These findings demonstrate that these insecticides modulate transcription
of GSTT and GSTA enzymes and may play an important role in the
modulation of endogenous compounds and xenobiotic metabolism. Studies are in
progress to define the molecular mechanisms by which chlorpyrifos induce the expression
of GSTT and GSTA.
389 IDENTIFICATION OF TRANSCRIPTIONAL
REGULATORS OF C. ELEGANS METALLOTHIONEIN
GENE EXPRESSION.
J. Hall and J. H. Freedman. NIEHS, Durham, NC.
The carcinogenic metal cadmium can induce various intracellular stresses. Analysis
of transcriptome data from multiple species indicate that cadmium exposure alters
the expression of hundreds of genes that are regulated by multiple signal transduction
pathways, many of which remain to be defined. In response to cadmium, cells
increase the expression of highly conserved, small, cysteine-rich metal-binding proteins
known as metallothioneins (MTs), which function in metal detoxification.
The nematode C. elegans has two MT genes: mtl-1 and mtl-2. To identify regulatory
factors and pathways that control metal-inducible mtl-1 transcription, integrated
transgenic strains of C. elegans containing GFP under the control of the 5’regulatory
region of mtl-1 were constructed; pmtl-1::GFP. Transgenic strains
constitutively express GFP in the pharynx and following cadmium exposure, express
GFP in the intestine. Using the pmtl-1::GFP strains, genes involved in various
stress response pathways were tested for their potential role in controlling mtl-
1 expression. Knockout of akt-1 or akt-2 did not affect GFP expression; however,
the knockout of both genes simultaneously increased expression. AKT-1 is a serine/threonine
kinase involved in the insulin signaling pathway and complexes with
AKT-2 to regulate transcription of downstream factors. PDK-1 directly interacts
with this complex and the knockout of pdk-1 resulted in an increase in expression.
Interestingly, mtl-1 transcription was not affected when other insulin signaling
pathway genes were knocked out. This suggests that PDK-1 and the AKT-1/2 complex
act independently of this pathway to control mtl-1 transcription. To identify
other transcriptional regulators, transcription factors involved in various MAPK
pathways were tested. Knockout of atf-7 which is involved in the JNK/p38 pathway
resulted in an increase in GFP expression. Pathway analysis and RT-PCR data
indicate that ATF-7 regulates cadmium-inducible MT transcription downstream of
PDK-1 and AKT-1/2 and that this regulation is independent of the insulin signaling
pathway.
390 ANILINE-INDUCED CELL CYCLE PROGRESSION OF
SPLENOCYTES: RESPONSE OF CYCLINS AND CYCLIN-
DEPENDENT KINASES IN G1/S AND G2/M PHASES.
J. Wang, G. Wang and M. Khan. University of Texas Medical Branch, Galveston, TX.
Aniline exposure is associated with toxicity to the spleen leading to splenomegaly,
hyperplasia, fibrosis, and a variety of sarcomas of the spleen on chronic exposure in
rats. In earlier studies, we have shown that aniline exposure leads to iron overload,
oxidative stress and activation of redox-sensitive transcription factors, which could
regulate various genes leading to a tumorigenic response in the spleen. We have also
shown that aniline exposure leads to enhanced expression of cyclins and cyclin-dependent
kinases (CDKs) in G1 phase of cell cycle. However, molecular mechanisms
SOT 2011 ANNUAL MEETING 83